The Effects And Regulatory Mechanisms Of AcSDKP On Intestinal Inflammation In Ulcerative Colitis

Background & aims:Ulcerative colitis(UC)is a chronic,recurrent inflammatory disease of the intestinal tract that mainly affects the colon,characterized by the continuous inflammation of the mucosa and the submucosa.The pathogenesis of UC remains unclear and may be related to the complex interaction between genetic predisposition,environmental factors,disturbed intestinal microbiota and dysregulated immune response.Currently,all the drugs available for clinical UC therapy have some limitations,thus it is still urgent to search for novel agents with higher safety and better efficacy.N-acetyl-serylaspartyl-lysyl-proline(AcSDKP)is an endogenous small molecule with multiple biological activities,such as anti-inflammation,anti-fibrosis and pro-angiogenesis.AcSDKP is mainly released from its precursor thymosin β4(Tβ4)through stepwise hydrolysis mediated by meprin-α and prolyl endopeptidase(PREP)and degraded by angiotensin converting enzyme(ACE).Numerous evidences suggest that AcSDKP plays a protective role in several cardiovascular and renal diseases.However,little is known about the functions of AcSDKP in gastrointestinal diseases.Herein,we sought to explore the potential effects of AcSDKP on the intestinal inflammation in UC and tried to provide new ideas for the treatment of UC.Methods:Clinical research: The expressions of Tβ4,meprin-α,PREP,and ACE were assessed in the inflamed and non-inflamed tissues of patients with active UC by Real-time PCR and immunohistochemical staining to analyze the possible trends of AcSDKP contents in the inflamed intestinal tissues of UC patients.In vitro experiments: Caco-2 cells were exposed to TNF-α at the dose of 10ng/ml,with AcSDKP in different concentrations.Real-time PCR and Western blot were utilized to examine the expression of proinflammatory cytokines and signaling molecules involved in MAPK pathway and NF-κB pathway,aiming at exploring the anti-inflammatory effects of AcSDKP in vitro.Animal experiments: enzyme immunoassay was performed to detect the basal AcSDKP content in the colon of PREP knockout(PREP-KO)mice,in order to verify the influence of PREP deficiency on the AcSDKP level.Next,the murine models of UC were induced in PREP-KO mice through administrating of dextran sulfate sodium(DSS).Weight loss,stool consistency and fecal blood were observed daily to assess the disease activity index(DAI)and colon length was measured at the end of modeling.Hematoxylin-eosin(HE)staining and immunohistochemistry were used to evaluate the severity of colon injury and the degree of inflammatory cell infiltration.The expression of pro-inflammatory cytokines and associated signaling pathways in the colon of mice were determined by Real-time PCR and Western blot.Finally,mini osmotic pumps were employed to infuse exogenous AcSDKP into mice intraperitoneally,at a dose of1600μg/kg/day.Taking advantage of DSS-induced colitis models,we would clarify the role of AcSDKP in murine colitis.Results:1.The expression of the precursor and proteinase involved in AcSDKP generation or degradation in the intestine of patients with active UCIn the inflamed tissues of patients with active UC,the expression levels of Tβ4 and meprin-α were downregulated,while PREP and ACE were expressed at similar levels to the non-inflamed tissues,indicating that the amount of AcSDKP,in the inflamed intestinal tissues of UC patients,might be significantly reduced.2.Effects of AcSDKP on inflammatory responses of Caco-2 cells induced by TNF-αIn the TNF-α-stimulated Caco-2 cells,AcSDKP not only suppressed the expression of inflammatory cytokines,but also inhibited the activation of MEK and ERK,without affecting the phosphorylation of JNK,p38,IKKα/β,and p65,indicating that AcSDKP could attenuate inflammatory responses of Caco-2 cells in part by inhibiting MEK-ERK signaling.3.Effects of AcSDKP on DSS-induced colitis and intestinal inflammatory environments in C57BL/6 micePREP deficiency significantly downregulated the basal AcSDKP content in the colon of mice,suggesting that PREP indeed plays an important role in the generation of AcSDKP.Upon DSS administration,PREP-KO mice exhibited more weight loss,higher DAI scores,shorter colon length,and greater colonic damage,compared with WT mice.Moreover,the expression levels of inflammatory cytokines were higher in the colonic tissues of PREP-KO mice.Additionally,more infiltrating inflammatory cells and activated MEK-ERK pathway were observed in PREP-KO mice.Collectively,these data indicate that PREP deficiency aggravates DSS-induced murine colitis.By contrast,among WT mice,AcSDKP treatment attenuated the symptoms of DSSinduced colitis and led to less colonic injury and weaker intestinal inflammation,demonstrating that AcSDKP has anti-inflammatory properties in the intestine.Conclusions:The expression of Tβ4 and meprin-α was decreased in the inflamed intestinal tissues of UC patients.In vitro,AcSDKP can inhibit TNF-α-induced inflammatory responses of intestinal epithelial cells,showing potent anti-inflammatory effects.In vivo,PREP deficiency accelerated the development of murine colitis,which may be partly attributed to the decreased level of colonic AcSDKP.In contrast,AcSDKP treatment can ameliorate murine colitis and indeed has protective effects on the intestine.